CN101862353B - Method for extracting total triterpenes in fungus larch - Google Patents
Method for extracting total triterpenes in fungus larch Download PDFInfo
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Abstract
The invention discloses a method for extracting total triterpenes in fungus larch, which comprises the following steps of: taking the fungus larch, and extracting the fungus larch by using an extracting solvent to obtain extract of the fungus larch, wherein the extracting solvent is 60-95 percent (volume percentage) ethanol water, the dosage of the extracting solvent is 8-12 times of the mass of the fungus larch, and the extracting temperature is 80-100 DEG C. Shown by results, the optimal extraction process of the total triterpenes in the fungus larch is as follows: refluxing and extracting with 80 percent alcohol two times by heating , extracting for 3h first time by adding 10 times of alcohol, and extracting for 1.5h second time by adding 8 times of alcohol, wherein the average of the contents of the total triterpenes in the extract reaches 84.03+/-0.77 percent, the average of extraction ratios reaches 22.45+/-0.38 percent, and the content of dehydrosulfurenic acid reaches 5.17+/-0.10 percent.
Description
Technical field
The present invention relates to total triterpene acid method for distilling in the Fructificatio Fomitopsis Officinalis.
Background technology
Fructificatio Fomitopsis Officinalis [Fomes officinialis (Vill.ex.Fr.) Ames] is the fungus sporophore that the Polyporaceae shelf fungus belongs to.The bitter white hoof of popular name claims that Fructificatio Fomitopsis Officinalis is " fungus of larch " in " Uygur medicine medicinal herbs most in use " book of Xinjiang dimension literary composition version, and Uighur is " Harry is female ".Profile is the shape of a hoof, and the edge is irregular, differs in size, and big diameter can reach 30～40cm, 5 little～10cm, and the sporophore outside is pale brown color or canescence, inner white.Record in Drug Standard of Ministry of Public Health of the Peoples Republic of China " Uigurs medicine fascicle ".Fructificatio Fomitopsis Officinalis is Xinjiang uighur ' s culture doctor's a common drug, and folk remedy is also in order to cure the disease.It is dry, hot that dimension doctor thinks that Fructificatio Fomitopsis Officinalis is gone into the property of medicine, has that stomach warming eliminates the phlegm, a function of lowering the adverse-rising QI to subdue asthma, expelling wind and removing dampness, swelling diuretic, is one of medical material of Uygur medicine treatment chronic tracheitis commonly used.Pharmacological evaluation shows that Fructificatio Fomitopsis Officinalis has warming the lung for resolving phlegm, sending down the abnormal ascending QI, promoting blood circulation and detumescence, diuresis.For one of main component of Uigurs medicine compound recipe Fructificatio Fomitopsis Officinalis sheet, be mainly used in eliminating phlegm and relieving asthma.
The foreign scholar shows that to the chemical constitution study of Fructificatio Fomitopsis Officinalis its main component is triterpenic acid, fatty acid etc., reports in " Uigurs medicine will " that it contains multiple triterpene, steroidal compounds, and wherein triterpenic acid content can be up to 16%.
Triterpenes components is one of effective ingredient of a lot of medicinal fungis; Medicinal fungi is being paid much attention to aspect raising immune function of human body, defying age and other the many health cares; Its kind is many, distribution is wide, breeding is fast; In contemporary Chinese medicine, playing an important role, is an important ingredient of Chinese herbal medicine.The triterpenic acid compounds has many researchs in the Fructificatio Fomitopsis Officinalis, but standardized Study on extraction of triterpenoid and content assaying method are not seen bibliographical information, makes the quality control of extract be difficult to realize, bioactive result of study, and also just becoming is difficult to certainly.Thereby we have designed three levels, four factor orthogonal tests, have carried out the standardization Study on Extraction Method of Fructificatio Fomitopsis Officinalis triterpenoid.
Summary of the invention
The object of the present invention is to provide a kind of preparation method of extract of Fructificatio Fomitopsis Officinalis.
The preparation method of extract of Fructificatio Fomitopsis Officinalis provided by the invention comprises the steps: to get Fructificatio Fomitopsis Officinalis, and said Fructificatio Fomitopsis Officinalis is extracted with the extraction solvent, collects extracting solution, obtains the extract of said Fructificatio Fomitopsis Officinalis;
Said extraction solvent is 60%-95% (percent by volume) ethanol water; The consumption of said extraction solvent is 8-12 a times of said Fructificatio Fomitopsis Officinalis quality; The temperature of said extraction is 80-100 ℃.
For abundant extraction, Fructificatio Fomitopsis Officinalis can be pulverized earlier before extracting, and crossed 10 mesh sieves.
The method of said extracted specifically can be a reflux, extract,, and the number of times of said reflux, extract, is 2-4 time, and each 1-4 hour, the temperature of said reflux, extract, was 80-100 ℃.
In one embodiment, the said extracted solvent can be the ethanol water of 95% (percent by volume); The consumption of said extraction solvent is 10 times of said Fructificatio Fomitopsis Officinalis quality; The temperature of said reflux, extract, is 100 ℃; The number of times of said reflux, extract, is 3 times, each 1-3 hour.
In another embodiment, the said extracted solvent is the ethanol water of 60-80% (percent by volume); The consumption of said extraction solvent is said Fructificatio Fomitopsis Officinalis quality 8-10 times; The temperature of said reflux, extract, is 80-100 ℃; The number of times of said reflux, extract, is 2 times, each 1.5-3 hour.
Further, the number of times of above-mentioned reflux, extract, is 2 times, and the consumption of the extraction solvent of the 1st reflux, extract, is 10 times of said Fructificatio Fomitopsis Officinalis quality, and the time of said the 1st reflux, extract, is 3 hours; The consumption of the extraction solvent of the 2nd reflux, extract, is 8 times of said Fructificatio Fomitopsis Officinalis quality, and the time of said the 2nd reflux, extract, is 1.5 hours; Said extraction solvent is the ethanol water of 60% or 70% or 80% (percent by volume); The temperature of said reflux, extract, is 100 ℃.
Said method also comprise with said extracting solution through concentrating under reduced pressure then vacuum drying obtain pressed powder to constant weight as the extract of said Fructificatio Fomitopsis Officinalis.
Further, above-mentioned method for preparing also comprises measures total triterpene acid content in the above-mentioned pressed powder, and its step is following:
A) in above-mentioned pressed powder, add 5% vanillin-glacial acetic acid and perchloric acid, close plug mixing, reaction is 20-30 minute under 50-70 ℃ of condition, places stopped reaction on the ice bath then rapidly, obtains chromophoric solution; Said 5% vanillin-glacial acetic acid is that the 5g vanillin is dissolved in the glacial acetic acid, and the reuse glacial acetic acid is settled to the solution that obtains behind the 100mL; Wherein the proportioning of pressed powder and 5% vanillin-glacial acetic acid and perchloric acid is (0.03-0.05) mg: (0.2-0.8) ml: (0.8-1.4) mL;
B) add the glacial acetic acid standardize solution in the chromophoric solution in the step a), survey absorbance, calculate total triterpene acid content in the extract that above-mentioned method prepares according to standard curve then in the 554nm wavelength; Wherein the proportioning of volume behind the standardize solution and the pressed powder in the step a) is 10mL: (0.03-0.05) mg.
In step a), the proportioning of said pressed powder and 5% vanillin-glacial acetic acid and perchloric acid is 0.04mg: 0.5mL: 1.0mL; Said reaction temperature is 60 ℃, and the response time is 25 minutes.
In order to prevent false-positive appearance, above-mentioned method for preparing also comprises measures dehydrogenation sulfurenic acid (dehydrosulphurenic acid) content in the above-mentioned pressed powder, and its step is following:
I) above-mentioned pressed powder is dissolved as solvent with methanol, obtain sample solution;
II) with step I) sample solution that obtains dilution back obtains filtrating with membrane filtration;
III) with Step II) filtrating that obtains carries out HPLC and measures, and according to dehydrogenation sulfurenic acid standard curve, calculates the dehydrogenation Polyporus sulphureus acid content in the above-mentioned pressed powder.
At step I) in, the proportioning of said methanol and said pressed powder is (0.04-0.06) g: (10-75) mL, optimum ratio are 0.05g: (10-75mL);
In Step II) in, the aperture of said filter membrane is 0.45 μ m; Step II I) in, said HPLC condition is a chromatographic column: the Zorbax Eclipse XDB-C of Agilent Technologies company
18(250mm * 4.6mm, 5 μ m) analytical column; Mobile phase: acetonitrile-0.4% phosphate aqueous solution (61: 39); Flow velocity: 1.0mL/min; Detect wavelength: 242nm; Column temperature: 25 ℃.
The orthogonal experiment Preliminary screening shows: 4 factors influences importance to it and are followed successively by extraction solvent (A)＞method for distilling (B)＞extraction time (D)＞extraction time (C), intuitive analysis overall merit R value, and optimum extraction process is A
2B
2C
3D
1, promptly 95% alcohol heating reflux extracts 3 times, each 1 hour.
Through the orthogonal experiment results of preliminary screening, and considering the safety of cost and reagent, select ethanol economy, safety the most for use, is that solvent makes further to select to carry out single factor analysis with ethanol.The result shows: the optimum extraction process of triterpenic acid constituents is that 80% alcohol heating reflux extracts twice in the Fructificatio Fomitopsis Officinalis; Add for the first time 10 times of amount ethanol extraction 3h; Add for the second time 8 times of amount ethanol extraction 1.5h; Total triterpene acid content meansigma methods reaches 84.03 ± 0.77% in the extract at this moment, and the extraction ratio meansigma methods reaches 22.45 ± 0.38 (%), and dehydrogenation Polyporus sulphureus acid content reaches 5.17 ± 0.10%.
Most of bibliographical informations to triterpene (acid) constituents method for distilling in other medical materials preferred in; Be that index is analyzed only to total triterpene contents or extraction ratio; Do not consider that extraction conditions is to wherein certain has the monomer component content of pharmacologically active; False positive results possibly occur like this, make the method that optimizes unreliable.It among the present invention the interference of avoiding this phenomenon; Measuring total triterpene acid content and considering that its extraction ratio simultaneously; Adopting wherein, characteristic chemical constituent dehydrogenation sulfurenic acid is a reference substance; Measure this kind triterpene monomer component content, carry out the analysis-by-synthesis evaluation, finally confirm the standardization method for distilling of triterpenic acid compounds in the Fructificatio Fomitopsis Officinalis.
Description of drawings
Fig. 1 dehydrogenation sulfurenic acid structural formula.
Fig. 2 reference substance dehydrogenation sulfurenic acid high-efficient liquid phase chromatogram.
Fig. 3 Fructificatio Fomitopsis Officinalis medical material need testing solution high-efficient liquid phase chromatogram.
Fig. 4 dehydrogenation sulfurenic acid reference substance is at 242nm wavelength uv absorption spectra.
When Fig. 5 test sample retention time is 9.774min at 242nm wavelength uv absorption spectra.
Fig. 6 dehydrogenation sulfurenic acid reference substance standard curve.
Figure 75 % vanillin-glacial acetic acid consumption is investigated the result.
Fig. 8 test sample and reference substance thin-layer chromatogram, 1. test sample I; 2. dehydrogenation sulfurenic acid reference substance; 3. test sample II; 4. dehydrogenation sulfurenic acid reference substance.
Fig. 9 dehydrogenation sulfurenic acid reference substance (A) and need testing solution (B) colour developing back visible spectrum scintigram.
Figure 10 dehydrogenation sulfurenic acid reference substance standard curve (total triterpene acid assay).
Need testing solution high-efficient liquid phase chromatogram during Fructificatio Fomitopsis Officinalis medical material water extraction in Figure 11 orthogonal experiment 1.
The high-efficient liquid phase chromatogram of Fructificatio Fomitopsis Officinalis medical material water extraction need testing solution in Figure 12 orthogonal experiment 2.
The high-efficient liquid phase chromatogram of Fructificatio Fomitopsis Officinalis medical material water extraction need testing solution in Figure 13 orthogonal experiment 3.
The high-efficient liquid phase chromatogram of Fructificatio Fomitopsis Officinalis medical material 95% ethanol extraction need testing solution in Figure 14 orthogonal experiment 4.
The high-efficient liquid phase chromatogram of Fructificatio Fomitopsis Officinalis medical material 95% ethanol extraction need testing solution in Figure 15 orthogonal experiment 5.
The high-efficient liquid phase chromatogram of Fructificatio Fomitopsis Officinalis medical material 95% ethanol extraction need testing solution in Figure 16 orthogonal experiment 6.
The high-efficient liquid phase chromatogram of Fructificatio Fomitopsis Officinalis medical material dichloromethane extraction need testing solution in Figure 17 orthogonal experiment 7.
The high-efficient liquid phase chromatogram of Fructificatio Fomitopsis Officinalis medical material dichloromethane extraction need testing solution in Figure 18 orthogonal experiment 8.
The high-efficient liquid phase chromatogram of Fructificatio Fomitopsis Officinalis medical material dichloromethane extraction need testing solution in Figure 19 orthogonal experiment 9.
The high-efficient liquid phase chromatogram of Figure 20 Fructificatio Fomitopsis Officinalis medical material 60% ethanol extraction need testing solution.
Need testing solution high-efficient liquid phase chromatogram during Figure 21 Fructificatio Fomitopsis Officinalis medical material 70% ethanol extraction.
Need testing solution high-efficient liquid phase chromatogram during Figure 22 Fructificatio Fomitopsis Officinalis medical material 80% ethanol extraction.
The specific embodiment
Below in conjunction with specific embodiment the present invention is described further, but the present invention is not limited to following examples.
Among the following embodiment,, be conventional method like no specified otherwise.
Fructificatio Fomitopsis Officinalis is purchased the Uygur medicine hospital in Xinjiang in October, 2007, is accredited as the sporophore that the Polyporaceae shelf fungus belongs to Fructificatio Fomitopsis Officinalis [Fomes officinialis (Vill.ex.Fr.) Ames] through the Liu Qinghua researcher of institute of materia medica, Xinjiang; For the ease of extracting the composition in the Fructificatio Fomitopsis Officinalis, in following examples, earlier Fructificatio Fomitopsis Officinalis is ground into coarse powder (crossing 10 mesh sieves).
Dehydrogenation sulfurenic acid reference substance (purity 99.5%) behind the purification, the structural formula of dehydrogenation sulfurenic acid is as shown in Figure 1.The method for preparing of the reference substance behind the purification wherein: the dry sporophore 5Kg of Fructificatio Fomitopsis Officinalis; Alcohol reflux with 95% 2 times; Merge extractive liquid, reclaim behind the solvent about 700 grams of extractum, extractum is dissolved in an amount of 95% the ethanol, with 100-200 order silica gel mixed sample; Successively with petroleum ether, chloroform, ethyl acetate, acetone and methanol-eluted fractions, chloroform extract 240g (Fr.B) wherein.Fr.B is through silica gel column chromatography, and chloroform-methanol (10: 0～6: 4～0: 10) gradient elution is divided into Fr.1～6 six part.Fr.3 part warp silica gel column chromatography (chloroform-methanol 10: 1～6: 4～0: 10) repeatedly obtains dehydrogenation Polyporus sulphureus acid crude; Dehydrogenation Polyporus sulphureus acid crude is dissolved in a small amount of pyridine; The C18 silica gel chromatographic column is through methanol-water (5: 5) 400mL balance; The wet method application of sample with methanol-water (6: 4～7: 3～8: 2) gradient elution, is collected each Concentraton gradient eluent.Receive eluting stream part by every part of 250mL, must flow part (1)-(11) respectively, (12)-(15), (16)-(24).With the small amount of methanol dissolving, carry out the thin layer inspection and know behind each stream part evaporated under reduced pressure solvent, developing solvent is chloroform-methanol (9: 1), and developer is 10% sulphuric acid.Compare according to thin layer chromatography speckle displacement and dehydrogenation sulfurenic acid reference substance; Manifest purple dot with reference substance in same position behind the point sample, and do not see the impurity speckle, be indicated as target compound; Its solvent is volatilized and vacuum drying; The white solid powder, be the reference substance behind the purification, the reference substance moderate purity behind the detection purification is 99.5%.
The foundation and the detection thereof of dehydrogenation sulfurenic acid content assaying method in embodiment 1, the Fructificatio Fomitopsis Officinalis
One, the foundation of dehydrogenation sulfurenic acid content assaying method in the Fructificatio Fomitopsis Officinalis
1, extracts choice of Solvent
Precision takes by weighing 6 parts of Fructificatio Fomitopsis Officinalis coarse powder, and each 2g splits in 6 50mL tool plug triangular flasks, adds 50mL methanol, 70% methanol, dehydrated alcohol, 70% ethanol, 50% ethanol, water respectively; Close plug is claimed to decide weight, and all ultrasonic (35kHz) extracts 1h, puts cold; Weigh again, supply the weight that subtracts mistake, shake up, filter with each solvent; Filtering residue more respectively once with solvent repetitive operation separately, merging filtrate respectively, evaporated under reduced pressure, residue changes with methanol and dissolves to the 25mL measuring bottle; Add methanol again and be diluted to scale, shake up, filter, get subsequent filtrate with 0.45 μ m microporous filter membrane; Promptly get the need testing solution of six kinds of solvent extraction gained, each sample introduction 5 μ L adopts HPLC to measure each peak area value, represents wherein dehydrogenation Polyporus sulphureus acid content with peak area.
Wherein the HPLC testing conditions is: chromatographic column: the Zorbax EclipseXDB-C of Agilent Technologies company
18(250mm * 4.6mm, 5 μ m) analytical column; Mobile phase: acetonitrile-0.4% phosphate aqueous solution (61: 39); Flow velocity: 1.0mL/min; Detect wavelength: 242nm; Column temperature: 25 ℃.
The result sees shown in the table 1: methanol is put forward the peak area maximum of solution dehydrogenation sulfurenic acid; Wherein dehydrogenation Polyporus sulphureus acid content is the highest; And filtration time is shorter relatively, and aqueous solution dehydrogenation Polyporus sulphureus acid content is minimum, so final particular methanol is the optimum extraction solvent of need testing solution preparation.
Table 1 different solvents extracts investigates the result
2, the selection of method for distilling
On step 1 has confirmed that methanol is for the basis of extracting solvent, following three groups of experiments are set confirm best method for distilling:
1) ultrasonic extraction: get the about 2g of Fructificatio Fomitopsis Officinalis medicinal material coarse powder, the accurate title, decide, and puts in the 100mL tool plug triangular flask, adds methanol 60mL, close plug; Claim to decide weight, ultrasonic (35kHz) extracts 1h, puts coldly, weighs again, supplies the weight that subtracts mistake with methanol; Shake up, filter, the filtering residue repetitive operation once, merging filtrate, concentrating under reduced pressure; The reuse methanol constant volume shakes up to the 25mL measuring bottle, filters with 0.45 μ m microporous filter membrane, gets subsequent filtrate, promptly gets need testing solution.
2) reflux extraction: get the about 2g of Fructificatio Fomitopsis Officinalis medicinal material coarse powder, the accurate title, decide, and places round-bottomed flask, adds methanol 60mL, claims to decide weight; Reflux 1h is put coldly, weighs again, supplies the weight that subtracts mistake with methanol, shakes up; Filter, the filtering residue repetitive operation once, merging filtrate, concentrating under reduced pressure; The reuse methanol constant volume shakes up to the 25mL measuring bottle, filters with 0.45 μ m microporous filter membrane, promptly gets need testing solution.
3) soxhlet extraction: get the about 2g of Fructificatio Fomitopsis Officinalis medicinal material coarse powder, the accurate title, decide, and places apparatus,Soxhlet's; Add methanol 70mL, Suo Shi extracts 3h, and extracting solution is evaporated in right amount; The reuse methanol constant volume shakes up to the 25mL measuring bottle, filters with 0.45 μ m microporous filter membrane; Get subsequent filtrate, promptly get need testing solution.
Each sample introduction 5 μ L of above need testing solution adopt HPLC (testing conditions is identical with step 1) to measure each peak area value, represent wherein dehydrogenation Polyporus sulphureus acid content with peak area.
Table 2 Different Extraction Method is investigated the result
The result is as shown in table 2, and ultrasonic method is the highest to the extraction ratio of dehydrogenation sulfurenic acid in the medical material, and the operating time is shorter relatively, and is comparatively simple and convenient, is the optimum extracting method of need testing solution preparation so select ultrasonic method.
3, the selection of extraction time
On the basis of step 1 and 2, the experiment of different extraction times is set:
Precision takes by weighing 3 parts of Fructificatio Fomitopsis Officinalis medicinal material coarse powder, and each 2g splits in the 100mL tool plug triangular flask, adds methanol 60mL, close plug; Claim decide weight, ultrasonic (35kHz) processing 20min, 40min, 60min are respectively put coldly, weigh again; Supply the weight that subtracts mistake with methanol, shake up, filter, filtrate decompression is concentrated near doing; The reuse methanol constant volume shakes up to the 25mL measuring bottle, filters with 0.45 μ m microporous filter membrane, gets subsequent filtrate; Promptly get the need testing solution of different extraction times, all each sample introduction 5 μ L adopts HPLC (testing conditions is identical with step 1) to measure each peak area value, represents wherein dehydrogenation Polyporus sulphureus acid content with peak area.
The different extraction times investigation of table 3 result
The result is as shown in table 3, and supersound extraction 60min can more fully extract the dehydrogenation sulfurenic acid in the Fructificatio Fomitopsis Officinalis medical material, so finally to select with methanol supersound extraction 60min be that need testing solution prepares optimum extracting method.
4, the best approach of dehydrogenation sulfurenic acid assay in the Fructificatio Fomitopsis Officinalis
1) preparation of reference substance solution
The accurate title, decided dehydrogenation sulfurenic acid 5.57mg, places the 10mL volumetric flask, adds dissolve with methanol, and reuse methanol is diluted to scale, shakes up, and promptly gets reference substance solution (every 1mL contains dehydrogenation sulfurenic acid reference substance 0.557mg).
2) preparation of need testing solution
Get the about 2g of Fructificatio Fomitopsis Officinalis medicinal material coarse powder, the accurate title, decide, and places 100mL tool plug triangular flask, adds methanol 60mL; Close plug is claimed to decide weight, and ultrasonic (35kHz) handles 60min, puts cold; Claim again to decide weight, supply the weight that subtracts mistake, shake up, filter with methanol; Filtrate decompression is concentrated near doing, and the reuse methanol constant volume shakes up to the 25mL measuring bottle.Therefrom accurate absorption 0.5mL places the 10mL measuring bottle,, with its dilution twice, filters with 0.45 μ m microporous filter membrane to scale with methanol constant volume again, gets subsequent filtrate, promptly gets need testing solution.
3) detect
Reference substance solution and step 2 with step 1)) each sample introduction 10 μ l of need testing solution, adopt HPLC (chromatographic condition is identical with step 1) to measure, record dehydrogenation sulfurenic acid peak area value, external standard method calculating content.
The high-efficient liquid phase chromatogram of reference substance solution and need testing solution is distinguished as shown in Figures 2 and 3, and the appearance time of dehydrogenation sulfurenic acid is respectively 9.871min and 9.833min.
Need testing solution when the retention time of reference substance solution and collection is 9.774 minutes at 242nm wavelength uv absorption spectra respectively like Fig. 4 and shown in Figure 5; Find that need testing solution and reference substance solution all have absorption maximum in the 242nm wavelength; And blank is noiseless, so the final detection wavelength of selecting 242nm as dehydrogenation sulfurenic acid assay in the Fructificatio Fomitopsis Officinalis.
Two, the detection of dehydrogenation sulfurenic acid content assaying method in the Fructificatio Fomitopsis Officinalis
1, the preparation of standard curve
The accurate respectively dehydrogenation sulfurenic acid reference substance solution of drawing in the step 1 (0.557mg/mL) 0.2,0.4,0.6; 0.8,1.0,1.2mL is placed in the 5mL measuring bottle; Add methanol respectively and be diluted to scale, shake up, promptly get serial dehydrogenation sulfurenic acid reference substance solution.Above-mentioned six concentration reference substance solution are sample introduction 10 μ l respectively; Adopting HPLC to measure each peak area value (result sees table 4), is abscissa with reference substance solution concentration, and peak area is a vertical coordinate; The drawing standard curve; Getting regression equation is y=6450.95x+5.48972, r=0.9997, and the result shows: the dehydrogenation sulfurenic acid is good linear relationship in the 0.0223-0.1337mg/ml scope.Standard curve is seen Fig. 6.
Table 4 dehydrogenation sulfurenic acid standard curve data determination result
2, precision test
Precision takes by weighing Fructificatio Fomitopsis Officinalis coarse powder 2g, processes need testing solution according to the method in the step 4 of step 1, carries out HPLC according to the chromatographic condition in the step 1 and detects.Repeat sample introduction 5 times, measure peak area.The result is as shown in table 5, and relative standard deviation (RSD) is 0.37% (n=5), and precision is good.
Table 5 Precision test result (n=5)
3, stability test
Precision takes by weighing Fructificatio Fomitopsis Officinalis coarse powder 2g, processes need testing solution according to the method in the step 4 of step 1, and respectively 0,2,4,6,8,12,24h sample introduction 10 μ l adopt HPLC to measure each peak area value.The result is as shown in table 6, and average peak area is 539.01, and its stable RSD is 1.80%, shows that need testing solution is basicly stable in 24h.
Table 6 stability test result (n=7)
4, replica test
Precision takes by weighing 5 parts of Fructificatio Fomitopsis Officinalis medicinal material coarse powder 2g respectively; According to 5 parts of need testing solutions of the preparation of the method in the step 4 of step 1, each sample introduction 10 μ l, the reference substance solution of going forward side by side 10 μ l; Adopt HPLC to measure each peak area value, calculate wherein dehydrogenation Polyporus sulphureus acid content with external standard method.The result is as shown in table 7, and the RSD of its method repeatability is 1.20% (n=5).
Table 7 replica test result (n=5)
5, average recovery test
It is 6 parts of 3.9124% Fructificatio Fomitopsis Officinalis medicinal material coarse powder that precision takes by weighing known dehydrogenation Polyporus sulphureus acid content, and every part of 0.025g adds a certain amount of dehydrogenation sulfurenic acid reference substance, adds methanol 20mL respectively, close plug; Claim to decide weight, ultrasonic (35kHz) handles 60min, puts coldly, weighs again; Supply the weight that subtracts mistake with methanol, shake up, filter, with methanol constant volume to the 25mL measuring bottle; Shake up, 0.45 μ m microporous filter membrane filters, and gets subsequent filtrate, promptly gets.Sample and reference substance solution be each sample introduction 10 μ l all, adopts HPLC to measure peak area value, calculates dehydrogenation Polyporus sulphureus acid content, and its average recovery average is 100.68%, RSD=1.97% (n=6).The result sees table 8.
Table 8 average recovery result of the test (n=6)
The foundation and the detection thereof of embodiment 2, Fructificatio Fomitopsis Officinalis total triterpene acid component content assay method
One, the foundation of Fructificatio Fomitopsis Officinalis total triterpene acid component content assay method
1, the selection of developer (5% vanillin-glacial acetic acid) consumption
The preparation of need testing solution: get the about 2g of Fructificatio Fomitopsis Officinalis medicinal material coarse powder, the accurate title, decide, and places cable type extractor according; Add dichloromethane 70mL, extract 2.5h, the extracting solution evaporated under reduced pressure; Residue is with dissolve with methanol and be settled in the 25mL measuring bottle, shakes up, and promptly gets the need testing solution of present embodiment.
Accurate again absorption 0.1mL need testing solution places the 25mL measuring bottle, is diluted to scale with methanol, shakes up.Therefrom accurate respectively absorption 0.2,0.5,0.8,1.1,1.4mL splits in the 10mL tool plug test tube; Water bath method takes out and puts to room temperature, the accurate respectively 5% vanillin-glacial acetic acid 0.5mL that adds, and perchloric acid 1.0mL shakes up; Close plug is put heated at constant temperature 20min in 60 ℃ of water-baths, takes out, and puts in the ice-water bath immediately and cools off, and is transferred in the 10mL measuring bottle; Add glacial acetic acid and be diluted to scale, shake up, the contrast of retinue blank solution is measured absorbance in the 554nm wavelength respectively.
Table 95% vanillin-glacial acetic acid consumption is investigated the result
Result such as table 9 increase along with sampling amount becomes multiple with shown in Figure 7, the proportional increase of absorbance, and linear relationship is good, r=0.9985.Proof 0.5mL vanillin-glacial acetic acid can with the triterpenic acid constituents complete reaction in the institute sample thief, and it is easy to operate accurately to take 0.5mL, so that the developer consumption is 0.5mL is comparatively suitable.
2, the selection of developer (perchloric acid) consumption
Precision takes by weighing Fructificatio Fomitopsis Officinalis medicinal material coarse powder 2g, prepares need testing solution by the method for the step 1 in the step 1 of present embodiment, accurately again draws 0.1mL and places the 25mL measuring bottle, is diluted to scale with methanol, shakes up.The accurate 0.5mL that draws, totally 6 parts, split in the 10mL tool plug test tube, water bath method takes out and puts to room temperature, the accurate respectively 5% vanillin-glacial acetic acid 0.5mL that adds; Be followed successively by perchloric acid 0.4,0.6,0.8,1.0,1.2 each accurate adding again; 1.4mL, mixing, close plug is put in 60 ℃ of water-baths and is heated 20min, takes out to put in the ice-water bath immediately and cools off; Be transferred in the 10mL measuring bottle, add glacial acetic acid and be diluted to scale, shake up, the contrast of retinue blank solution is measured absorbance in the 554nm wavelength respectively.
Table 10 perchloric acid consumption consumption is investigated the result
The result is as shown in table 10, and along with the rising of perchloric acid consumption, absorbance increases, and absorbance is maximum when reaching 1.0mL, and after this increase its consumption absorbance again and reduce, be the perchloric acid optimum amount so select 1.0mL.
3, the selection of bath temperature (reaction temperature)
Precision takes by weighing Fructificatio Fomitopsis Officinalis medicinal material coarse powder 2g, prepares need testing solution by the method for the step 1 in the step 1 of present embodiment, accurately again draws 0.1mL and places the 25mL measuring bottle, is diluted to scale with methanol, shakes up.The accurate 0.5mL that draws, totally 6 parts, split in the 10mL tool plug test tube, water bath method takes out and puts to room temperature, the accurate respectively 5% vanillin-glacial acetic acid 0.5mL that adds; Perchloric acid 1.0mL, mixing, close plug places 40,50,60 respectively; React 20min in 70,80,90 ℃ of water-baths, take out, put in the ice-water bath immediately and cool off; Be transferred in the 10mL measuring bottle, add glacial acetic acid and be diluted to scale, shake up, the contrast of retinue blank solution is measured absorbance in the 554nm wavelength respectively.
Table 11 different temperatures is investigated the result
The result is as shown in table 11, and with the rising of bath temperature, absorbance increases, and relatively stable at 50-70 ℃ of scope internal absorbance in 40-90 ℃ of scope, so select 60 ℃ of bath temperatures when developing the color.
4, the selection of developing time
Precision takes by weighing the about 2g of Fructificatio Fomitopsis Officinalis medicinal material coarse powder, prepares need testing solution by the method for the step 1 in the step 1 of present embodiment, accurately again draws 0.1mL and places the 25mL measuring bottle, is diluted to scale with methanol, shakes up.The accurate 0.5mL that draws, totally 6 parts, split in the 10mL tool plug test tube, water bath method takes out and puts to room temperature, the accurate respectively 5% vanillin-glacial acetic acid 0.5mL that adds; Perchloric acid 1.0mL, mixing, close plug places 60 ℃ of water-baths respectively, heats 5,10 successively; 15,20,25,30min takes out, and puts in the ice-water bath immediately and cools off; Be transferred in the 10mL measuring bottle, add glacial acetic acid and be diluted to scale, shake up, the contrast of retinue blank solution is measured absorbance in the 554nm wavelength respectively.
The different developing times of table 12 are investigated the result
The result is as shown in table 12, and in the 5-25min scope, with the colour developing prolongation of heat time heating time, absorbance increases, and absorbance descends after surpassing 25min, and relatively stable at 20-30min scope internal absorbance, so select 25min as developing time.
Finally select with 5% vanillin-glacial acetic acid 0.5mL, perchloric acid 1.0mL, 60 ℃ of heating colour developings 25 minutes were the best coloration method of total triterpene acid composition in the Fructificatio Fomitopsis Officinalis.
5, dichloromethane is as the feasibility analysis of extracting solvent
Precision takes by weighing Fructificatio Fomitopsis Officinalis medicinal material coarse powder 2.0053g, prepares need testing solution by the method for the step 1 in the step 1 of present embodiment.
Precision takes by weighing dehydrogenation sulfurenic acid reference substance 2.02mg, places the 10mL measuring bottle, adds that an amount of methanol is ultrasonic to make dissolving, adds methanol again and is diluted to scale, shakes up, and promptly gets the dehydrogenation sulfurenic acid reference substance solution of 0.202mg/mL.
Draw test sample and dehydrogenation sulfurenic acid reference substance solution (0.202mg/mL) respectively, point sample is developing solvent with chloroform-methanol (9: 1) on same silica gel g thin-layer plate, launches, and takes out, and dries.Spray is with 10% sulfuric acid solution, and it is clear to be heated to the speckle colour developing, on test sample and the corresponding position of dehydrogenation sulfurenic acid reference substance solution thin layer chromatography, and apparent identical purple dot.Put under the ultra-violet lamp (365nm) and inspect, the fluorescence speckle all do not occur in test sample and the reference substance chromatograph.Show that making solvent with dichloromethane can more fully extract triterpenic acid constituents in the Fructificatio Fomitopsis Officinalis.Its thin layer chromatography is seen accompanying drawing 8.
6, detect the selection of wavelength
Get the about 2g of Fructificatio Fomitopsis Officinalis medicinal material coarse powder, accurate claim surely, prepare need testing solution by the method for the step 1 in the step 1 of present embodiment; Accurate again absorption 0.1mL places the 25mL measuring bottle; The accurate reference substance solution 2mL that draws places the 10mL measuring bottle, is diluted to scale with methanol, shakes up.Accurate need testing solution 0.5mL and the reference substance solution 1.0mL that draws after diluting splits in the 10mL tool plug test tube, and water bath method takes out and puts to room temperature respectively; The accurate respectively 5% vanillin-glacial acetic acid 0.5mL that adds, perchloric acid 1.0mL shakes up, close plug; Put heated at constant temperature 20min in 60 ℃ of water-baths, take out, put in the ice-water bath immediately and cool off; Be transferred in the 10mL measuring bottle, add glacial acetic acid and be diluted to scale, shake up.The contrast of retinue blank solution; Respectively in the interscan of 400～800nm wave-length coverage; Find that need testing solution and reference substance solution all have absorption maximum in the 554nm wavelength, and blank is noiseless, so the final detection wavelength of selecting 554nm as Fructificatio Fomitopsis Officinalis total triterpene acid constituents assay.Its visible spectrum scintigram is seen Fig. 9.
Two, the detection of Fructificatio Fomitopsis Officinalis total triterpene acid component content assay method
1, the preparation of standard curve
Precision takes by weighing dehydrogenation sulfurenic acid reference substance 2.02mg, places the 10mL measuring bottle, adds that an amount of methanol is ultrasonic to make dissolving, adds methanol again and is diluted to scale, shakes up, and promptly gets.Accurate respectively again absorption 0.2,0.4,0.6,0.8,1.0; 1.2mL place 6 10mL tool plug test tubes (table 13 numbering is respectively 1,2,3,4,5 and 6), water bath method takes out and puts to room temperature, accurate respectively 5% vanillin-glacial acetic acid 0.5mL, the perchloric acid 1.0mL of adding; Mixing, close plug is put heated at constant temperature 25min in 60 ℃ of water-baths, takes out; Put in the ice-water bath immediately and cool off, be transferred in the 10mL measuring bottle, add glacial acetic acid and be diluted to scale, shake up.Directly do not add 5% vanillin-glacial acetic acid 0.5mL not add need testing solution; Perchloric acid 1.0mL uses glacial acetic acid to be diluted to graduated mixed solution and is blank, surveys absorbance (its result sees table 13) in the 554nm wavelength respectively; With the reference substance solution concentration C is abscissa; Absorbance A is a vertical coordinate, the drawing standard curve, and getting regression equation is A=23.84C+0.0297; R=0.9989, the result shows: dehydrogenation sulfurenic acid standard substance are good in 0.0040-0.0242mg/ml scope internal linear relation.Standard curve is seen Figure 10.
Table 13 dehydrogenation sulfurenic acid standard curve data determination result
2, precision test
Get and be numbered 4 the reference substance solution after colour developing in the above-mentioned steps 1, survey its absorbance continuously 5 times in the 554nm wavelength, the result sees table 14, and RSD is 0.17% (n=5), and precision is good.
Table 14 Precision test result (n=5)
3, replica test
Get each about 2g of 5 parts of Fructificatio Fomitopsis Officinalis medicinal material coarse powder respectively, accurate claim surely, prepare need testing solution by the method for the step 1 in the step 1 of present embodiment, the accurate 0.1mL that draws places the 25mL measuring bottle, and the therefrom accurate again 0.5mL that draws splits in the 10mL tool plug test tube.Water bath method takes out and puts to room temperature, accurate respectively 5% vanillin-glacial acetic acid 0.5mL, the perchloric acid 1.0mL of adding; Mixing, close plug is put heated at constant temperature 25min in 60 ℃ of water-baths, takes out; Put in the ice-water bath immediately and cool off, be transferred in the 10mL measuring bottle, add glacial acetic acid and be diluted to scale, shake up.Directly do not add 5% vanillin-glacial acetic acid 0.5mL not add need testing solution; Perchloric acid 1.0mL uses glacial acetic acid to be diluted to graduated mixed solution and is blank, surveys absorbance in the 554nm wavelength respectively; Retinue dehydrogenation sulfurenic acid reference substance carries out the absorbance test; Calculate total triterpene acid content in the Fructificatio Fomitopsis Officinalis, the result sees table 15, and the RSD of its method repeatability is 2.82% (n=5).
Table 15 replica test result (n=5)
4, stability test
Get the about 2g of Fructificatio Fomitopsis Officinalis medicinal material coarse powder, accurate claim surely, prepare need testing solution, accurately again draw 0.1mL and place the 25mL measuring bottle by the method for the step 1 in the step 1 of present embodiment.Water bath method takes out and puts to room temperature, accurate respectively 5% vanillin-glacial acetic acid 0.5mL, the perchloric acid 1.0mL of adding; Mixing, close plug is put heated at constant temperature 25min in 60 ℃ of water-baths, takes out; Put in the ice-water bath immediately and cool off, be transferred in the 10mL measuring bottle, add glacial acetic acid and be diluted to scale, shake up.Directly do not add 5% vanillin-glacial acetic acid 0.5mL not add need testing solution; Perchloric acid 1.0mL uses glacial acetic acid to be diluted to graduated mixed solution and is blank, surveys absorbance in the 554nm wavelength respectively; Retinue dehydrogenation sulfurenic acid reference substance carries out the absorbance test; In the time of 0,10,20,30,60,90,120 and 150 minute, wavelength 554nm place surveys absorbance respectively, and the result sees table 16.
Table 16 stability test result
The result shows in the Fructificatio Fomitopsis Officinalis that the total triterpene acid composition is after developing the color in the 60min; Absorbance changes less, and RSD is 2.59%, and RSD rises to 3.32% during 90min; Along with time lengthening; Absorbance constantly descends, and should after colour developing, accomplish in the 60min so this method is measured, and stability of solution is better during this.
5, average recovery test
Precision takes by weighing 6 parts of the Fructificatio Fomitopsis Officinalis coarse powder of known content, and every part of about 0.004g adds a certain amount of dehydrogenation sulfurenic acid reference substance, prepares need testing solution by the method for the step 1 in the step 1 of present embodiment; Water bath method takes out and puts to room temperature, accurate respectively 5% vanillin-glacial acetic acid 0.5mL, the perchloric acid 1.0mL of adding; Mixing, close plug is put heated at constant temperature 25min in 60 ℃ of water-baths, takes out; Put in the ice-water bath immediately and cool off, be transferred in the 10mL measuring bottle, add glacial acetic acid and be diluted to scale, shake up.Directly do not add 5% vanillin-glacial acetic acid 0.5mL not add need testing solution, perchloric acid 1.0mL uses glacial acetic acid to be diluted to graduated mixed solution and is blank; Survey absorbance in the 554nm wavelength respectively; Retinue dehydrogenation sulfurenic acid reference substance carries out the absorbance test, surveys absorbance at wavelength 554nm place, calculates total triterpene acid content in the Fructificatio Fomitopsis Officinalis; Its average recovery average is 98.56%, RSD=1.89% (n=6).The result sees table 17.
Table 17 average recovery result of the test (n=6)
The standardization Study on Extraction Method of triterpenic acid composition in embodiment 3, the Fructificatio Fomitopsis Officinalis
One, carries out the research of orthogonal test to extracting solvent, method for distilling, extraction time and extraction time
1, the preparation of dry extract
Get the Fructificatio Fomitopsis Officinalis medical material, be ground into coarse powder (crossing 10 mesh sieves), precision takes by weighing totally 27 parts, and every part of about 5g extracts by the orthogonal experiment design of table 19, and each sample repeats to extract operation 3 times.All add 10 times of each solvents of amount at every turn, claim to decide weight, supply the weight that subtracts mistake, shake up,, 3 extracting solution are merged concentrating under reduced pressure, put in the evaporating dish that is dried to constant weight, carry out vacuum drying (0.09MPa) collecting extracting solution after the extract coarse filtration with each solvent.The dry 12h of 95% ethanol extraction wherein, the dry 5h of dichloromethane extract, more than the dry 40h of water extract, pressed powder, weigh respectively, can try to achieve dry extract powder amount.
Each factor level in the orthogonal test scheme of its invading the exterior 19 is shown in table 18, adopts conventional four factors, three horizontal quadrature design methods, selects L for use
9(3
4) orthogonal design table; To extract solvent, method for distilling, extraction time and extraction time is empirical factor; Each factor is established 3 levels, is the Preliminary screening that index is carried out the extraction process condition with the extraction ratio and the dehydrogenation Polyporus sulphureus acid content of total triterpene acid in the medical material.
Table 18 orthogonal experiment factor and water-glass
Table 19 orthogonal experiment design
2, the mensuration of total triterpene acid component content in the Fructificatio Fomitopsis Officinalis
Precision takes by weighing each extract dry extract powder respectively, and every part of about 0.01g puts in the 25mL measuring bottle, uses an amount of dissolve with methanol, adds methanol again and is diluted to scale, shakes up.
Extract is if water extract then, and the accurate 0.5mL that draws splits in the 10mL tool plug test tube water bath method from the 25mL measuring bottle; Taking-up is put to room temperature, accurate respectively 5% vanillin-glacial acetic acid 0.5mL, perchloric acid 1.0mL, the mixing of adding; Close plug is put heated at constant temperature 25min in 60 ℃ of water-baths, takes out, and puts in the ice-water bath immediately and cools off; Be transferred in the 10mL measuring bottle, add glacial acetic acid and be diluted to scale, shake up.Directly do not add 5% vanillin-glacial acetic acid 0.5mL not add need testing solution, perchloric acid 1.0mL uses glacial acetic acid to be diluted to graduated mixed solution and is blank, surveys absorbance in the 554nm wavelength respectively.
Like berry extract is extractive with organic solvent, and the difference of itself and water extract is: before the water bath method step, and the accurate 2mL that draws from the 25mL measuring bottle earlier; Put in the 10mL measuring bottle; Add methanol and be diluted to scale, shake up, the accurate again 0.5mL that draws; Split in the 10mL tool plug test tube, afterwards operation and the same water extract of assay method.
With absorbance substitution regression equation, calculate total triterpene acid content and extraction ratio in each sample.Orthogonal experiment results is seen table 20, and table 21 is seen in variance analysis.
Behind orthogonal experiment extraction and assay; Intuitive analysis result (seeing table 20) shows: with total triterpene acid extraction ratio in the Fructificatio Fomitopsis Officinalis is index; 4 factors influence importance to it and are followed successively by and extract solvent (A)＞method for distilling (B)＞extraction time (D)＞extraction time (C), and calculating its optimum extraction process by intuitive analysis is A
2B
2C
3D
1, promptly 95% alcohol heating reflux extracts 3 times, each 1 hour.The results of analysis of variance (seeing table 21) shows that type of solvent has the influence (P＜0.01) of highly significant to extraction ratio, and the influence of other factors is not remarkable.Variance analysis can get R
2=0.992 (R of adjustment
2=0.966), explains that this multifactor Tobin's mean variance model is higher to the overall fitting degree of total triterpene acid extraction ratio data.
Table 20 Orthogonal experiment results and analysis (I)
Total triterpene acid content (%)=(total triterpene acid quality in the dry extract/take dry extract quality) * 100%; Extraction ratio (%)=[(the total dry extract quality of total triterpene acid content * gained)/take Fructificatio Fomitopsis Officinalis quality of medicinal material] * 100%
Table 21 variance analysis (I)
F
0.10(2，2)＝9.0?F
0.05(2，2)＝19.0?F
0.01(2，2)＝99.0
3, dehydrogenation sulfurenic acid Determination on content
Get the extract dry extract powder that has made, every part of about 0.05g of water extract, accurate respectively title is fixed, puts in the 5mL measuring bottle, uses an amount of dissolve with methanol, and is settled to scale, shakes up.Every part of about 0.01g of 95% ethanol extraction, accurate respectively title is fixed, places the 25mL measuring bottle, uses an amount of dissolve with methanol, and is settled to scale, shakes up.Every part of about 0.05g of dichloromethane extract respectively accurately claims surely, puts in the 10mL measuring bottle, with dissolve with methanol and be settled to scale, shakes up, and therefrom accurately again draws 5mL in the 10mL measuring bottle, adds methanol constant volume to scale, shakes up.Each solution all filters with 0.45 μ m microporous filter membrane, gets subsequent filtrate, promptly gets need testing solution.
Carry out HPLC according to the chromatographic condition among the embodiment 1 and detect sample size 10 μ L.The high-efficient liquid phase chromatogram of each solvent Different Extraction Method need testing solution such as Figure 11-shown in Figure 19.According to peak area value and reference substance dehydrogenation sulfurenic acid equation of linear regression, calculate triterpene monomer component content in each sample.The result sees table 22.
Table 22 Orthogonal experiment results (II)
Dehydrogenation Polyporus sulphureus acid content (%)=(dehydrogenation sulfurenic acid quality in the dry extract/take dry extract quality) * 100%;
4, the analysis-by-synthesis of Orthogonal experiment results
The pair of orthogonal experimental result adopts integrated evaluating method, and with total triterpene acid extraction ratio weight coefficient 0.7, dehydrogenation Polyporus sulphureus acid content weight coefficient 0.3 is as comprehensive evaluation index.
Overall merit score value=total triterpene acid extraction ratio * 0.7+ dehydrogenation Polyporus sulphureus acid content * 0.3.The result sees table 23.
Press comprehensive evaluation index, table 23 result shows: 4 factors influences importance to it and are followed successively by extraction solvent (A)＞method for distilling (B)＞extraction time (D)＞extraction time (C), intuitive analysis overall merit R value, and optimum extraction process is A
2B
2C
3D
1, promptly 95% alcohol heating reflux extracts 3 times, each 1 hour.Variance analysis (seeing table 24) shows that type of solvent has the influence (P＜0.01) of highly significant to experimental result, and the influence of other factors is not remarkable.Variance analysis can get R
2=0.992 (R of adjustment
2=0.969), explains that this multifactor Tobin's mean variance model is higher to the overall fitting degree of triterpenic acid constituents overall merit data.
Table 23 Orthogonal experiment results and analysis (III)
Table 24 variance analysis (II)
F
0.10(2，2)＝9.0?F
0.05(2，2)＝19.0?F
0.01(2，2)＝99.0
Two, Different concentrations of alcohol is as the investigation of extracting solvent
Consider that actual production saves time and the cost problem, carry out experiment of single factor, investigate the influence of different concentration ethanol total triterpene acid content, extraction ratio and dehydrogenation Polyporus sulphureus acid content in the Fructificatio Fomitopsis Officinalis.For ease of experimental design and commercial production, extraction time is chosen to be twice by conventional method for distilling.
1, the preparation of dry extract
Get the Fructificatio Fomitopsis Officinalis medical material, be ground into coarse powder (crossing 10 mesh sieves), precision takes by weighing totally 9 parts, every part of about 5g.Extract with 60%, 70% and 80% alcohol heating reflux respectively and add 10 times of amount ethanol for twice, the 1 time, extract 3h, add 8 times of amount ethanol for the second time, extract 1.5h, each sample repeats to extract operation 3 times.When extracting at every turn, 9 duplicate samples, shake up all in solvent to the former weight of putting cold back replenish lost.With collecting extracting solution after the extract coarse filtration; 3 extracting solution are merged concentrating under reduced pressure, put in the evaporating dish that is dried to constant weight, vacuum drying (0.09MPa); 60%, the dry 30h of 70% and 80% ethanol extraction difference, 22h, 16h; Get pressed powder, weigh respectively, can try to achieve dry extract powder weight.
2, the mensuration of total triterpene acid component content
Precision takes by weighing extract dry extract powder respectively, and every part of about 0.01g puts in the 25mL measuring bottle, with an amount of dissolve with methanol and be settled to scale, shakes up.The therefrom accurate 2mL that draws puts in the l0mL measuring bottle, adds methanol and is diluted to scale, shakes up; The accurate again 0.5mL that draws splits in the 10mL tool plug test tube, and water bath method takes out and puts to room temperature; The accurate respectively 5% vanillin-glacial acetic acid 0.5mL that adds, and perchloric acid 1.0mL (proportioning that is powder and 5% vanillin-glacial acetic acid and perchloric acid is 0.04mg: 0.5ml: 1.0ml), and mixing, close plug; Put heated at constant temperature 25min in 60 ℃ of water-baths, take out, put in the ice-water bath immediately and cool off; Be transferred in the 10mL measuring bottle, add glacial acetic acid and be diluted to scale, shake up.Directly do not add 5% vanillin-glacial acetic acid 0.5mL not add need testing solution; Perchloric acid 1.0mL; Use glacial acetic acid to be diluted to graduated mixed solution and be blank, survey absorbance in the 554nm wavelength respectively, utilize total triterpene acid content and extraction ratio in each sample of regression equation calculation.The result sees table 25.
Table 25 different concentration ethanol influences result (I) (n=3) to what extract
Can find out the influence of extraction ratio according to different concentration ethanol in Orthogonal experiment results in the table 20 and analysis and the table 25; Extraction ratio meansigma methods when using 80% ethanol is 22.4874% (RSD=1.69%; N=3); Close with extraction ratio maximum (23.4006%) in the orthogonal experiment, this method is stable, feasible.
3, dehydrogenation sulfurenic acid Determination on content
Get the extract dry extract powder that has made, 60% ethanol extraction, 70% ethanol extraction, every part of about respectively 0.05g of 80% ethanol extraction, the accurate title, decide, and puts in the 10mL measuring bottle, uses an amount of dissolve with methanol, and be settled to scale, shakes up.Wherein 70% ethanol extraction is therefrom accurate again draws 5mL in the 10mL measuring bottle, adds methanol constant volume to scale, shakes up.80% ethanol extraction is therefrom accurate draws 2mL in the 10mL measuring bottle, adds methanol constant volume to scale, shakes up.Each solution all filters with 0.45 μ m microporous filter membrane, gets subsequent filtrate, promptly gets need testing solution.
Carry out HPLC according to the chromatographic condition among the embodiment 1 and detect sample size 10 μ L.60%, the need testing solution high-efficient liquid phase chromatogram of 70%, 80% ethanol extraction such as Figure 20-shown in Figure 22.According to peak area value and reference substance dehydrogenation sulfurenic acid equation of linear regression, calculate dehydrogenation Polyporus sulphureus acid content and extraction ratio in each sample.The result sees table 26.
Table 26 different concentration ethanol influences result (II) (n=3) to what extract
Can calculate according to result in table 25 and the table 26 and the integrated evaluating method in the present embodiment, the overall merit score value is 17.2902 when using 80% ethanol, and approaching with maximum in the orthogonal experiment (18.1349), method is feasible.Consider the influence of total triterpene acid extraction ratio and index components dehydrogenation Polyporus sulphureus acid content simultaneously; So confirming the optimum extraction process of triterpenic acid constituents in the Fructificatio Fomitopsis Officinalis is that 80% alcohol heating reflux extracts twice; Add for the first time 10 times of amount ethanol extraction 3h, add 8 times of amount ethanol extraction 1.5h for the second time.
Organic solvent extraction efficient is apparently higher than water extraction in the leaching process, and 80% ethanol extraction can reduce water-solubility impurity, helps further separation and purification, and solvent is nontoxic, residual, recyclable recycles, and production cost is lower.This extraction process extraction ratio is higher, in the hope of becoming the standardization method for distilling of triterpenes components in the Fructificatio Fomitopsis Officinalis, for screening Fructificatio Fomitopsis Officinalis pharmacologically active provides reliable and stable sample preparation methods.
Claims (4)
1. the detection method of a Fructificatio Fomitopsis Officinalis extract comprises the steps: to get Fructificatio Fomitopsis Officinalis, and said Fructificatio Fomitopsis Officinalis is extracted with the extraction solvent, collects extracting solution, obtains the extract of said Fructificatio Fomitopsis Officinalis;
The method of said extraction is a reflux, extract; The number of times of said reflux, extract, is 2 times, and the consumption of the extraction solvent of the 1st reflux, extract, is 10 times of said Fructificatio Fomitopsis Officinalis quality, and the time of said the 1st reflux, extract, is 3 hours; The consumption of the extraction solvent of the 2nd reflux, extract, is 8 times of said Fructificatio Fomitopsis Officinalis quality, and the time of said the 2nd reflux, extract, is 1.5 hours; Said extraction solvent is the ethanol water of 80% (percent by volume); The temperature of said extraction is 80-100 ℃;
With said extracting solution through concentrating under reduced pressure then vacuum drying obtain pressed powder to constant weight as the extract of said Fructificatio Fomitopsis Officinalis;
Said detection method comprises measures total triterpene acid content in the described pressed powder, and its step is following:
A) in described pressed powder, add 5% vanillin-glacial acetic acid and perchloric acid, close plug mixing, reaction is 20-30 minute under 50-70 ℃ of condition, places stopped reaction on the ice bath then rapidly, obtains chromophoric solution; Said 5% vanillin-glacial acetic acid is that the 5g vanillin is dissolved in the glacial acetic acid, and the reuse glacial acetic acid is settled to the solution that obtains behind the 100mL; Wherein the proportioning of pressed powder and 5% vanillin-glacial acetic acid and perchloric acid is (0.03-0.05) mg: (0.2-0.8) mL: (0.8-1.4) mL;
B) add the glacial acetic acid standardize solution in the chromophoric solution in the step a), survey absorbance in the 554nm wavelength then, calculate total triterpene acid content in the extract for preparing according to standard curve then; Wherein the proportioning of volume behind the standardize solution and the pressed powder in the step a) is 10mL: (0.03-0.05) mg;
Said detection method also comprises measures dehydrogenation Polyporus sulphureus acid content in the said pressed powder, and its step is following:
I) described pressed powder is dissolved as solvent with methanol, obtain sample solution;
II) with step I) sample solution that obtains dilution back obtains filtrating with membrane filtration;
III) with Step II) filtrating that obtains carries out HPLC and measures, and according to dehydrogenation sulfurenic acid standard curve, calculates the dehydrogenation Polyporus sulphureus acid content in the described pressed powder;
Said HPLC condition is a chromatographic column: the Zorbax Eclipse XDB-C of Agilent Technologies company
18Analytical column, 250mm * 4.6mm, 5 μ m; Mobile phase: acetonitrile: 0.4% phosphate aqueous solution=61: 39; Flow velocity: 1.0mL/min; Detect wavelength: 242nm; Column temperature: 25 ℃.
2. the method for claim 1, it is characterized in that: in the step a), the proportioning of said pressed powder and 5% vanillin-glacial acetic acid and perchloric acid is 0.04mg: 0.5mL: 1.0mL; Said reaction temperature is 60 ℃, and the response time is 25 minutes.
3. method as claimed in claim 2 is characterized in that: step I), the proportioning of said methanol and said pressed powder is (0.04-0.06) g: (10-75) mL.
4. method as claimed in claim 3 is characterized in that: Step II), the aperture of said filter membrane is 0.45 μ m.
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